A first glycoside hydrolase family 50 endo-ß-1,3-d-glucanase from Pseudomonas aeruginosa.

A novel ß-1,3-glucanase gene (PaBglu50A) from Pseudomonas aeruginosa CAU 342A was cloned and expressed in Escherichia coli. The deduced amino acid sequence of PaBglu50A showed the highest identity of 34% with the ß-agarase belonging to glycoside hydrolase (GH) family 50. The purified PaBglu50A had maximal activity at pH 5.5 and 45°C, respectively. It was stable in the range of pH 4.0-8.0 and at temperatures below 40°C. The Km and Vmax of PaBglu50A for curdlan and laminarin were 94.4mgml-1 and 23.4µmolmin-1mg-1, 3.65mgml-1 and 8.89µmolmin-1mg-1, respectively. All characterized members of GH family 50 were only active towards agarose so far. However, the recombinant protein PaBglu50A did not display activity towards agarose but showed activity towards water-insoluble curdlan and laminarin. The hydrolysis products for curdlan supported this protein to be an endo-ß-1,3-glucanase, making a significant difference from the reported enzymes of GH family 50. These results suggested that PaBglu50A is the first endo-type ß-1,3-glucanase (EC 3.2.1.39) in GH family 50.

Enzymatic and molecular characterization of an endo-1,3-ß-d-glucanase from the crystalline styles of the mussel Perna viridis.

The retaining endo-1,3-ß-d-glucanase (EC 3.2.1.39) was isolated from the crystalline styles of the commercially available Vietnamese edible mussel Perna viridis. It catalyzes hydrolysis of ß-1,3-bonds in glucans and enables to catalyze a transglycosylation reaction. Resources of mass-spectrometry for analysis of enzymatic products were studied. cDNA sequence of endo-1,3-ß-d-glucanase was determined by RT-PCR in conjunction with the rapid amplification of cDNA ends (RACE) methods. The cDNA of 1380bp contains an open reading frame of 1332bp encoding a mature protein of 328 amino acids. On basis of amino acid sequence analysis endo-1,3-ß-d-glucanase was classified as a glycoside hydrolase of family 16.

Identification of the catalytic residues of the first family of beta(1-3)glucanosyltransferases identified in fungi.

A new family of glycosylphosphatidylinositol-anchored beta(1-3)glucanosyltransferases (Gelp), recently identified and characterized in the filamentous fungus Aspergillus fumigatus, showed functional similarity to the Gas/Phr/Epd protein families, which are involved in yeast morphogenesis. Sequence comparisons and hydrophobic cluster analysis (HCA) showed that all the Gas/Phr/Epd/Gel proteins belong to a new family of glycosylhydrolases, family 72. We confirmed by site-directed mutagenesis and biochemical analysis that the two conserved glutamate residues (the putative catalytic residues of this family, as determined by HCA) are involved in the active site of this family of glycosylhydrolases.